Gamma secretase substrates and in vitro assays

ABSTRACT

The present invention features γ-secretase substrates and in vitro assays for measuring γ-secretase activity employing such substrates. The γ-secretase substrates described herein contain a hydrophilic polypeptide moiety covalently joined to the carboxyl terminus of a β-CTF domain. A “β-CTF domain” is a polypeptide that can be cleaved by γ-secretase and which approximates the C-terminal fragment (amino acids 596-695) of APP produced after cleavage of APP by a β-secretase, or is a functional derivative thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to provisionalapplication U.S. Serial No. 60/201,053, filed May 1, 2000, herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] The references cited herein are not admitted to be prior art tothe claimed invention.

[0003] Amyloid precursor protein (APP) is a ubiquitous membrane-spanning(type 1) glycoprotein that undergoes a variety of proteolytic processingevents. (Selkoe, 1998. Trends in Cell Biology 8, 447-453.) Sequentialcleavage of APP by the β- and γ-secretases generate the N- andC-termini, respectively, of Aβ peptides which comprise amyloid plaquesin the brain parenchyma of patients with Alzheimer's disease. APPproteolysis by α-secretase occurs within the Aβ peptide domain therebyprecluding formation of the amyloidogenic peptides. The C-termini of theAβ peptides are heterogeneous. Peptides of 40 or 42 amino acids inlength (Aβ40 and Aβ42, respectively) are typically generated. Aβ42 ismore prone to aggregation than Aβ40 and is the major component ofamyloid plaque. (Jarrett et al., 1993. Biochemistry 32, 4693-4697; andKuo et al., 1996. J. Biol. Chem. 271, 4077-4081.)

[0004] The scissile bond for cleavage of APP by γ-secretase appears tobe situated within a transmembrane domain. It is unclear as to whetherthe C-termini of Aβ40 and Aβ42 are generated by a single protease withsloppy specificity or two distinct proteases. Recent studies suggestthat β-secretase also cleaves within the transmembrane region of Notchthereby releasing the Notch intracellular domain which controls crucialcell fate decisions during development. (De Strooper et al., 1999.Nature 398, 518-522.)

SUMMARY OF THE INVENTION

[0005] The present invention features γ-secretase substrates and invitro assays for measuring γ-secretase activity employing suchsubstrates. The γ-secretase substrates described herein contain ahydrophilic polypeptide moiety covalently joined to the carboxylterminus of a β-CTF domain.

[0006] A “β-CTF domain” is a polypeptide that can be cleaved byγ-secretase and which approximates the C-terminal fragment (amino acids596-695) of APP produced after cleavage of APP by a β-secretase, or is afunctional derivative thereof. Preferably, the β-CTF domain can becleaved by a γ-secretase to produce a fragment between about 39 to about43 amino acids in length. The preferred size ranges takes into accountthe generation of peptides Aβ40 and Aβ42 from naturally occurring APP bythe sequential actions of β-secretase and γ-secretase.

[0007] The hydrophilic polypeptide moiety is preferably chosen toincrease the solubility of the γ-secretase substrate in a zwitterionicdetergent. Hydrophilic moieties can be obtained taking into account theknown charges and polarity of different amino acid R groups.

[0008] Thus, a first aspect of the present invention features aγ-secretase substrate. The γ-secretase substrate contains a hydrophilicpolypeptide moiety covalently joined to the carboxyl terminus of a β-CTFdomain.

[0009] Another aspect of the present invention describes a nucleic acidcomprising a nucleotide base sequence encoding a γ-secretase substrate.Preferably, the nucleic acid is an expression vector.

[0010] Another aspect of the present invention describes a recombinantcell comprising a nucleic acid encoding a γ-secretase substrate.

[0011] Another aspect of the present invention describes a method forassaying γ-secretase activity comprising the use of an effective amountof a zwitterionic detergent and a γ-secretase substrate. γ-Secretaseactivity can be obtained from cells producing γ-secretase in asolubilized form or in a membrane-bound form. The effective amount of azwitterionic detergent is a concentration of zwitterionic detergentwhere γ-secretase produces detectable cleavage of the γ-secretasesubstrate.

[0012] The method can be performed by measuring product formationresulting from γ-secretase substrate cleavage. Measuring can beperformed by qualitative or quantitative techniques.

[0013] Another aspect of the present invention describes a method formeasuring the ability of a compound to affect γ-secretase activitycomprising the steps of: (a) combining together a γ-secretase substrate,a compound, and a preparation comprising γ-secretase activity, underreaction conditions allowing for γ-secretase activity, and (b) measuringγ-secretase activity. The reaction conditions allowing for γ-secretaseactivity comprise an effective amount of a zwitterionic detergent.

[0014] Other features and advantages of the present invention areapparent from the additional descriptions provided herein including thedifferent examples. The provided examples illustrate differentcomponents and methodology useful in practicing the present invention.The examples do not limit the claimed invention. Based on the presentdisclosure the skilled artisan can identify and employ other componentsand methodology useful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 provides a schematic representation of a fusion proteinconsisting sequentially of an N-terminal Met (M), APP597-695 and theFlag tag (Flag) sequence, and its processing by γ-secretase. The Aβ40-and Aβ42-related products (M-Aβ40 and M-Aβ42, respectively) are detectedby electrochemiluminescence (ECL) using biotinylated 4G8 antibody andruthenylated G2-10 or FCA3542, respectively.

[0016] FIGS. 2A-2D illustrate results of experiments characterizingdetergent-solubilized γ-secretase activity. (A) Dependence of M-Aβ40formation on the substrate concentration. The data show the ECL signalafter a 90 minute incubation at 37° C. (B) Time dependence of M-Aβ40formation by “solubilized γ-secretase”. The ECL signals are shown. (C)pH dependence of “solubilized γ-secretase” activity scoring forgeneration of the M-Aβ40. The ECL signals are shown. (D) Inhibition ofin vitro γ-secretase activity by pepstatin. The impact of pepstatin onthe generation of M-Aβ40 (∘) and M-Aβ42 () is shown. The ECL data isexpressed as % of activity observed in the absence of pepstatin. FIGS.2A, 2B and 2C show the mean values from two independent experiments.FIG. 2D shows the mean±SD (n=5).

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention features γ-secretase substrates and assaysfor detecting γ-secretase activity employing such substrates. Theγ-secretase substrate can be cleaved by γ-secretase activity.

[0018] Assaying for γ-secretase activity can be used, for example, topurify the enzyme, to characterize the enzyme, to screen for compoundsable to modulate γ-secretase activity, and to test the ability of aparticular compound to affect γ-secretase activity. Examples ofcompounds able to modulate γ-secretase activity include γ-secretaseinhibitors. Inhibitors can be employed for different purposes, such asin the treatment of Alzheimer's disease or characterization of thebiological importance of γ-secretase.

[0019] γ-Secretase Substrate

[0020] The γ-secretase substrate is a fusion protein comprising a β-CTFdomain and a hydrophilic polypeptide moiety. The β-CTF domain provides apolypeptide that can be cleaved by γ-secretase activity. The hydrophilicpolypeptide moiety allows for the β-CTF domain to be cleaved bydetergent-solubilized γ-secretase by promoting substrate solubility.

[0021] The β-CTF domain approximates the C-terminal fragment of APPafter cleavage by β-secretase or is a functional derivative thereof. Byapproximating the β-CTF portion of APP the γ-secretase substrate takesinto account the cleavage of APP by α- or β-secretase appearing to be aprerequisite for γ-secretase-mediated processing.

[0022] A “functional derivative thereof” has a sufficient sequencesimilarity to the β-CTF portion of APP such that it can be cleaved byγ-secretase. Examples of modifications to a β-CTF portion of APP toproduce a functional derivative include additions, deletions, andsubstitutions. The effect of a particular modification can be measuredusing reaction conditions described herein that allow for γ-secretasecleavage of a γ-secretase substrate. Preferred modifications do notcause a substantial decrease in activity. Preferably, additions anddeletions if present are located at the 5′ or 3′ end rather than beinginternal.

[0023] A “substantial decrease in activity” occurs when the observedactivity of γ-secretase is decreased 10 fold or more compared toactivity observed using a SEQ. ID. NO. 9 substrate incubated with cellmembranes or detergent-solubilized γ-secretase in the presence of 0.25%CHAPSO in buffer B (50 mM PIPES, pH 7.0, 5 mM MgCl₂, 5 mM CaCl₂, 150 mMKCl) at 37° C. (described in Example 4, infra.). In differentembodiments there is less than at a 5 fold or 2 fold decrease inactivity.

[0024] Substitutions in the substrate not causing a substantial decreasein activity can be initially designed taking into account differences innaturally occurring amino acid R groups. An R group affects differentproperties of the amino acid such as physical size, charge andhydrophobicity. Amino acids can be divided into different groups asfollows: neutral and hydrophobic (alanine, valine, leucine, isoleucine,proline, tryptophan, phenylalanine, and methionine); neutral and polar(glycine, serine, threonine, tyrosine, cysteine, asparagine, andglutamine); basic (lysine, arginine, and histidine); and acidic(aspartic acid and glutamic acid).

[0025] Generally, in substituting different amino acids it is preferableto exchange amino acids having similar properties. Substitutingdifferent amino acids within a particular group, such as substitutingvaline for leucine, arginine for lysine, and asparagine for glutamineare good candidates for not causing a change in polypeptide functioning.

[0026] Changes outside of different amino acids groups can also be made.Preferably, such changes are made taking into account the position ofthe amino acid to be substituted in the polypeptide. For example,arginine can substitute more freely for nonpolar amino acids in theinterior of a polypeptide then glutamate because of its long aliphaticside chain. (See, Ausubel, Current Protocols in Molecular Biology, JohnWiley, 1987-1998, Supplement 33 Appendix 1C.)

[0027] SEQ. ID. NO. 1 provides an example of a β-CTF domain. SEQ. ID.NO. 1 is a naturally occurring sequence corresponding to the β-CTFportion of APP (amino acids 596-695) along with an N-terminusmethionine. The N-terminus methionine facilitates recombinant productionof the substrate. SEQ. ID. NO. 1 is as follows: SEQ.ID.NO.1MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFF EQMQN.

[0028] Derivatives of the β-CTF portion of APP provided in SEQ. ID. NO.1 that are able to be cleaved in vivo by γ-secretase are well known inthe art. (See, for example, Lichtenthaler et al., 1997. Biochemistry 36,15396-15403; and Selkoe, 1999. Nature 399:A23-A31, which is not admittedto be prior art to the claimed invention.) Such derivatives canthemselves provide a β-CTF domain or can serve as a starting point forcreating additional derivatives.

[0029] Examples of naturally occurring derivatives of SEQ. ID. NO. 1 areprovided by SEQ. ID. NOs. 2-7. SEQ. ID. NOs. 2-7 correspond to the β-CTFportion of APP (amino acids 596-695) and also contain an N-terminusmethionine. SEQ. ID. NOs. 2-7, where differences between these sequencesand SEQ. ID. NO. 1 are highlighted, are provided as follows:SEQ.ID.NO.2: MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV VIATV VVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENP TYKFFEQMQN; SEQ.ID.NO.3:MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV VIATVI IITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENP TYKFFEQMQN; SEQ.ID.NO.4:MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV VIATVI GITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENP TYKFFEQMQN; SEQ.ID.NO.5:MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV VIATVI FITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENP TYKFFEQMQN; SEQ.ID.NO.6:MDAEFRHDSGYEVHHQKLVFF G EDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENP TYKFFEQMQN;SEQ.ID.NO.7: MDAEFRHDSGYEVHHQKLVFFA Q DVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENP TYKFFEQMQN.

[0030] In an embodiment of the present invention, the β-CTF domainsequence comprises, consists essentially of, or consists of, apolypeptide substantially similar to SEQ. ID. NO. 1. Preferably, theβ-CTF domain sequence comprises, consists essentially of, or consistsof, a sequence selected from the group consisting of SEQ.ID.NO.1,SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.6, andSEQ.ID.NO.7.

[0031] “Substantially similar” indicates a sequence similarity of atleast about 80% to a reference sequence. In different embodiments thesequence similarity is at least about 90%, at least about 95% or 100%.Sequence similarity can be determined using techniques well known in theart, such as those described by Altschul et al., 1997. Nucleic AcidsRes. 25, 3389-3402, hereby incorporated by reference herein. In oneembodiment sequence similarity is determined using tBLASTn searchprogram with the following parameters: MATRIX:BLOSUM62, PER RESIDUE GAPCOST: 11, and Lambda ratio: 1.

[0032] “Consists essentially” indicates that the reference sequence canbe modified by N-terminal and/or C-terminal additions or deletions thatdo not cause a substantial decrease in the ability of the γ-secretasesubstrate to be cleaved compared to the reference sequence. Preferably,additions or deletions if present are less than 5 amino acids on eitherend. An example of a deletion is the removal of the N-terminalmethionine.

[0033] A substantial decrease in the ability of the γ-secretasesubstrate to be cleaved is a decrease of 10 fold or more compared toactivity observed using a reference substrate incubated with cellmembranes or detergent-solubilized γ-secretase in the presence of 0.25%CHAPSO in buffer B (50 mM PIPES, pH 7.0, 5 mM MgCl₂, 5 mM CaCl₂, 150 mMKCl) at 37° C. (described in Example 4, infra.). In differentembodiments there is less than a 5 fold or 2 fold decrease in activity.

[0034] The second component of the γ-secretase substrate, thehydrophilic polypeptide moiety, is preferably chosen to increase thesolubility of the γ-secretase substrate in a zwitterionic detergent.Hydrophilic moieties can obtained taking into account the known chargesand polarity of different amino acid R groups. Preferably, the presenceof the hydrophilic moiety does not result in a substrate having asubstantial decrease in activity.

[0035] Different embodiments concerning the overall length and charge ofthe hydrophilic moiety are provided as follows: in different embodimentsconcerning the length, the length is about 5 to about 20 amino acids,about 8 to about 12 amino acids, or about 8 amino acids; in differentembodiments concerning the overall charge, the charge is greater than±2, ±3, or ±4. With respect to a negative charge, a greater chargeindicates a higher negative charge value.

[0036] Preferably, the hydrophilic moiety comprises, consistsessentially of, or consists of, a polypeptide substantially identical toSEQ. ID. NO. 8: DYKDDDDK. Substantially identical to SEQ. ID. NO. 8indicates that within a corresponding 8 amino acid stretch (no gaps)there is a two, one, or zero amino acid difference. Preferably, thehydrophilic moiety consists of the amino acid sequence of SEQ. ID. NO.8.

[0037] In an embodiment of the present invention concerning the overallstructure of the γ-secretase substrate, the γ-secretase substratecomprises, consists essentially of, or consists of, a sequencesubstantially similar to SEQ. ID. NO. 9. SEQ. ID. NO. 9 corresponds toSEQ. ID. NO. 1 along with a carboxyl terminal SEQ. ID. NO. 8 sequence.Preferably, the γ-secretase substrate comprises, consists essentiallyof, or consists of, a sequence selected from the group consisting of:SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12,SEQ.ID.NO.13,SEQ.ID.NO.14, and SEQ.ID.NO.15.

[0038] SEQ. ID. NOs. 9-15 are provided as follows (differences betweenSEQ. ID. NO. 9 and SEQ. ID. NOs. 10-15 are noted in bold andunderlined): SEQ.ID.NO.9 MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENP TYKFFEQMQNDYKDDDDK;SEQ.ID.NO.10 MDAEFRHDSGYEVHHQKLVFFAEDVGSNKG AIIGLMVGGVVIATV VVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLS KMQQNGYENPTYKFFEQMQNDYKDDDDK;SEQ.ID.NO.11 MDAEFRHDSGYEVHHQKLVFFAEDVGSNKG AIIGLMVGGVVIATVI IITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLS KMQQNGYENPTYKFFEQMQNDYKDDDDK;SEQ.ID.NO.12 MDAEFRHDSGYEVHHQKLVFFAEDVGSNKG AIIGLMVGGVVIATVI GITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLS KMQQNGYENPTYKFFEQMQNDYKDDDDK;SEQ.ID.NO.13 MDAEFRHDSGYEVHHQKLVFFAEDVGSNKG AIIGLMVGGVVIATVI FITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLS KMQQNGYENPTYKFFEQMQNDYKDDDDK;SEQ.ID.NO.14 MDAEFRHDSGYEVHHQKLVFF G EDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNDYKDDDDK; SEQ.ID.NO.15 MDAEFRHDSGYEVHHQKLVFFA QDVGSNKG AIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNDYKDDDDK.

[0039] Based on the disclosure provided herein γ-secretase substratescan be produced using standard biochemical synthesis and recombinantnucleic acid techniques. Techniques for chemical synthesis ofpolypeptides are well known in the art. (See, for example, Vincent, inPeptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990.)

[0040] Recombinant synthesis techniques for polypeptides are also wellknown in the art. Such techniques employ a nucleic acid template forpolypeptide synthesis. Starting with a particular amino acid sequenceand the known degeneracy of the genetic code, a large number ofdifferent encoding nucleic acid sequences can be obtained. Thedegeneracy of the genetic code arises because almost all amino acids areencoded by different combinations of nucleotide triplets or “codons”.The translation of a particular codon into a particular amino acid iswell known in the art (see, e.g., Lewin GENES IV, p. 119, OxfordUniversity Press, 1990).

[0041] Amino acids arc encoded for by codons as follows:

[0042] A=Ala=Alanine: codons GCA, GCC, GCG, GCU

[0043] C=Cys=Cysteine: codons UGC, UGU

[0044] D=Asp=Aspartic acid: codons GAC, GAU

[0045] E=Glu=Glutamic acid: codons GAA, GAG

[0046] F=Phe=Phenylalanine: codons UUC, UUU

[0047] G=Gly=Glycine: codons GGA, GGC, GGG, GGU

[0048] H=His=Histidine: codons CAC, CAU

[0049] I=Ile=Isoleucine: codons AUA, AUC, AUU

[0050] K=Lys=Lysine: codons AAA, AAG

[0051] L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

[0052] M=Met=Methionine: codon AUG

[0053] N=Asn=Asparagine: codons AAC, AAU

[0054] P=Pro=Proline: codons CCA, CCC, CCG, CCU

[0055] Q=Gln=Glutamine: codons CAA, CAG

[0056] R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

[0057] S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

[0058] T=Thr=Threonine: codons ACA, ACC, ACG, ACU

[0059] V=Val=Valine: codons GUA, GUC, GUG, GUU

[0060] W=Trp=Tryptophan: codon UGG

[0061] Y=Tyr=Tyrosine: codons UAC, UAU

[0062] Recombinant synthesis of polypeptides is achieved in a host cellusing an expression vector. An expression vector contains recombinantnucleic acid encoding for a desired polypeptide along with regulatoryelements for proper transcription and processing. The regulatoryelements that may be present include those naturally associated with therecombinant nucleic acid and exogenous regulatory elements not naturallyassociated with the recombinant nucleic acid. Exogenous regulatoryelements such as an exogenous promoter can be useful for expressingrecombinant nucleic acid in a particular host.

[0063] Generally, the regulatory elements that are present in anexpression vector include a transcriptional promoter, a ribosome bindingsite, a terminator, and an optionally present operator. A preferredelement is a polyadenylation signal providing for processing ineukaryotic cells. Other preferred elements include an origin ofreplication for autonomous replication in a host cell, a selectablemarker, a limited number of useful restriction enzyme sites, and apotential for high copy number. Examples of expression vectors arecloning vectors, modified cloning vectors, specifically designedplasmids and viruses.

[0064] Nucleic acid encoding for a polypeptide can be expressed in acell without the use of an expression vector employing, for example,synthetic mRNA or native mRNA. Additionally, mRNA can be translated invarious cell-free systems such as wheat germ extracts and reticulocyteextracts, as well as in cell based systems, such as frog oocytes.Introduction of mRNA into cell based systems can be achieved, forexample, by microinjection.

[0065] Techniques for introducing nucleic acid into an appropriateenvironment for expression, for expressing the nucleic acid to produceprotein, and for isolating expressed proteins are well known in the art.Examples of such techniques are provided in references such as Ausubel,Current Protocols in Molecular Biology, John Wiley, 1987-1998, andSambrook et al., in Molecular Cloning, A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor Laboratory Press, 1989.

[0066] γ-Secretase Assay

[0067] The γ-secretase substrate can be employed in assays measuringmembrane-bound or detergent-solubilized γ-secretase. Production ofcleavage products can be detected by Aβ peptide or hydrophilic moietyproduct formation.

[0068] Solubilized γ-secretase can be obtained from cells producingγ-secretase. Recovery of soluble γ-secretase activity is achieved usinga zwitterionic detergent during membrane extraction. Preferably, theamount of zwitterionic detergent is about 1% to about 2%. Examples ofcells producing γ-secretase include HeLa S3, human embryonic kidney(HEK293) cells and Chinese hamster ovary (CHO) cells. Examples ofzwitterionic detergents include CHAPSO and CHAPS.

[0069] Assay conditions employing membrane-bound ordetergent-solubilized γ-secretase allow for detectable γ-secretaseactivity. Such conditions include an effective amount of a zwitterionicdetergent, a buffer, and an appropriate temperature.

[0070] An effective amount of a particular zwitterionic detergentresults in detectable cleavage. Suitable detergents and amounts can bedetermined by evaluating the effect of a particular detergent onγ-secretase activity. Preferred zwitterionic detergents present duringthe assay are CHAPS and CHAPSO. A preferred percentage of suchdetergents is about 0.1% to about 0.5%.

[0071] An example of a reaction condition allowing for γ-secretaseactivity is provided as follows: 1.7 μM substrate incubated with cellmembranes or detergent solubilized γ-secretase in the presence of 0.25%CHAPSO in buffer B (50 mM PIPES, pH 7.0, 5 mM MgCl₂, 5 mM CaCl₂, 150 mMKCl) at 37° C. (described in Example 4, infra.). Such conditions can beused as a standard to determine effects of different components. Basedon the present disclosure such reaction conditions can be altered toprovide a wide range of additional reaction conditions allowing forγ-secretase activity. Preferably, changes to the reaction conditions donot result in a substantial decrease in activity.

[0072] γ-Secretase activity can be stopped using techniques well knownin the art for stopping enzymatic reactions. Preferably, γ-secretaseactivity is stopped using reagents compatible with subsequent analysis.

[0073] Cleavage of γ-secretase substrates can be measured by detectingformation of an Aβ type product or a product containing the hydrophilicmoiety. The presence of either of these products can be measured usingtechniques such as those employing antibodies and radioactive,electrochemiluminescent or fluorescent labels. If needed or desirable, apurification step enriching the different products may be employed.Examples of purification steps include the use of antibodies, separationgels, and columns.

[0074] Preferably, cleavage of γ-secretase is assayed for by detectingthe presence of Aβ-40 or Aβ-42. FIG. 1 illustrates a preferred methodfor product detection employing electrochemiluminescence with a captureantibody and an antibody specific for either Aβ-40 or Aβ-42. The captureantibody is used to enrich the products and hence, produce a highersignal.

EXAMPLES

[0075] Examples are provided below to further illustrate differentfeatures and advantages of the present invention. The examples alsoillustrate useful methodology for practicing the invention. Theseexamples do not limit the claimed invention.

Example 1

[0076] Recombinant Production of the γ-Secretase Substrate

[0077] A DNA fragment encoding amino acids 596-695 of the 695 amino acidisoform of APP (APP695) and SEQ. ID. NO. 8 at the C-terminus wasgenerated by PCR amplification of APP695 cDNA using appropriate primers.The employed primers had the following sequences: SEQ.ID.NO.16ggaattccatATGGATGCAGAATTCCGACATG; AND SEQ.ID.NO.17cgcggatccCTAtttatcgtcatcgtctttgtagtcGTTCTGCATCTGCT CAAAGAACTTG.

[0078] The Met that serves as the translation start site is residue 596of APP695 (the P1 residue with respect to the β-secretase cleavagesite). This DNA fragment was inserted into the procaryotic expressionvector pET2-21b (Novagen, Madison Wis.). The recombinant protein of SEQ.ID. NO. 9 was overproduced in E. coli [strain BL21(DE3)] and purified byMono-Q column chromatography (Pharmacia Biotech).

[0079] SEQ. ID. NO. 18 provides a nucleic acid sequence encoding for therecombinant protein of SEQ. ID. NO. 9 along with a stop codon.SEQ.ID.NO.18 ATGGATGCAGAATTCCGACATGACTCAGGATATGAAGTTCATCATCAAAAATTGGTGTTCTTTGCAGAAGATGTGGGTTCAAACAAAGGTGCAATCATTGGACTCATGGTGGGCGGTGTTGTCATAGCGACAGTGATCGTCATCACCTTGGTGATGCTGAAGAAGAAACAGTACACATCCATTCATCATGGTGTGGTGGAGGTTGACGCCGCTGTCACCCCAGAGGAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAACgactacaaagacgatgacgataaaTAG

Example 2

[0080] Aβ Peptide Detection

[0081] The Aβ peptides were detected using a sandwich assay employing anantibody to capture the peptide and an antibody to detect the presenceof the peptide. Detection was achieved by using ECL (Yang et al., 1994.Bio/Technology 12, 193-194; Khorkova et al., 1998. Journal ofNeuroscience Methods 82, 159-166), and an Origen 1.5 Analyzer (IgenInc., Gaithersburg, Md.).

[0082] Capture was performed using the 4G8 murine monoclonal antibody(Senetek PLC, Maryland Heights, Mo.). The 4G8 murine monoclonal antibodybinds an epitope in the Aβ peptide (within amino acids 18-21) that isimmediately distal to the α-secretase cleavage site. The 4G8 monoclonalantibody was biotinylated with Biotin-LC-Sulfo-NHS-Ester (Igen Inc.).

[0083] Detection was achieved using the G2-10 murine monoclonal antibodyand the FCA3542 rabbit antibody. The G2-10 murine monoclonal antibody(provided by K. Beyreuther, University of Heidelberg, Germany) binds theC-terminus that is exposed after γ-secretase-mediated cleavage togenerate amino acid 40 of the Aβ40 peptide. (Ida et al., 1996. J. Biol.Chem. 271, 22908-22914). The FCA3542 rabbit antibody (provided by F.Checler, IPMC du CNRS, Valbonne, France) binds the C-terminus that isexposed after γ-secretase-mediated cleavage to generate amino acid 42 ofthe Aβ42 peptide. (Barelli et al., 1997. Molecular Medicine 3, 695-707.)

[0084] The G2-10 and FCA3542 antibodies were ruthenylated with TAG-NHSEster (Igen Inc.). Aβ(x-40) was detected with biotinylated 4G8 andruthenylated G2-10. Aβ(x-42) was detected with biotinylated 4G8 andruthenylated FCA3542.

Example 3

[0085] Membrane Preparation and Detergent Solubilization

[0086] HeLa S3 cells from American Type Culture Collection (Rockville,Md.) were grown in bioreactors (Analytical Biological Services;Wilmington, Del.) in 90% DMEM, 10% fetal bovine serum, 2 mM glutamineand 100 μg/ml each of penicillin and streptomycin. Frozen HeLa S3 cellswere resuspended in buffer A (50 mM MES, pH 6.0, 5 mM MgCl₂, 5 mM CaCl₂,150 mM KCl) containing “complete” protease inhibitor cocktail(Boehringer Mannheim, Indianapolis, Ind.). The cells were broken bysingle-pass through a French Press (Spectronic Instruments, Rochester,N.Y.). Cell debris and nuclei were removed by centrifugation at 800×gfor 10 minutes. The supernatant solutions were centrifuged at 100,000×gfor 60 minutes. The ensuing pellets were resuspended in buffer A and thecentrifugation was repeated. The final membrane pellets were resuspendedin buffer A to yield a protein concentration of approximately 12 mg/ml.All procedures were performed at 4° C. The membranes were stored at −70°C.

[0087] HeLa cell membranes were treated with varying amounts of CHAPSO(up to 2.0%) followed by centrifugation. The supernatant solutions(solubilized fractions) and the pellets (membrane fractions) were thenassayed for γ-secretase activity with the SEQ. ID. NO. 9 substrate.

[0088] More γ-secretase activity is recovered in the solubilizedfraction, relative to the detergent-extracted membrane fraction, as theCHAPSO concentration is raised stepwise to 1.0% (Table I). The amountsof γ-secretase activity that are solubilized by 1.0% and 2.0% CHAPSO arecomparable. There are corresponding decreases in pellet-associatedγ-secretase activity as a result of the CHAPSO extraction. TABLE IExtraction of γ-secretase activity with CHAPSO detergent CHAPSOconcentration γ-secretase 0% 0.25% 0.50% 1.00% 2.0% preparationSolubilized <1 3 35 49 50 Membranes >99 97 65 51 50

[0089] Standard detergent solubilization of HeLa cell membranes (proteinconcentration, 2.5 mg/ml in buffer A) involved treatment with 1% CHAPSOfor 60 minutes at 4° C. and centrifugation at 100,000×g for 60 minutes.The ensuing supernatant solution provides “solubilized γ-secretase”.Approximately 50% of the γ-secretase activity in the HeLa cell membranesis solubilized by this CHAPSO extraction protocol. The total recovery ofγ-secretase activity in the “solubilized γ-secretase” and residualpellet is approximately 50% greater than that observed with the intactmembranes (data not shown).

Example 4

[0090] In Vitro γ-Secretase Assay

[0091] In vitro assays measuring γ-secretase activity were performedusing cell membranes or as “solubilized γ-secretase”. In one reaction,SEQ. ID. NO. 9 substrate (1.7 μM) was incubated with cell membranes (0.5mg/ml) in presence of detergent in buffer B (50 mM PIPES, pH 7.0, 5 mMMgCl₂, 5 mM CaCl₂, 150 mM KCl) at 37° C. In another reaction,supernatant solution from CHAPSO-extracted HeLa cell membranes(“solubilized γ-secretase”) was incubated with SEQ. ID. NO. 9 substrateat 37° C. in the presence of detergent in buffer B. Generally, 0.25%CHAPSO was provided as the detergent. The reactions were stopped byadding RIPA (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM TrisHCl, pH 8.0).

[0092] The samples were centrifuged and the supernatant solutions wereassayed for the Aβ peptides by ECL. The Aβ40- and Aβ42-related productsfrom γ-secretase-mediated processing of SEQ. ID. NO. 9 substrate possessa Met at the N-terminus and are thus defined as M-Aβ40 and M-Aβ42,respectively.

Example 5

[0093] Inhibition Studies

[0094] Inhibition studies was performed to demonstrate that theγ-secretase activity in “solubilized γ-secretase” is catalyzed by thebona fide APP processing enzyme in cells and is not simply due to aspurious proteolytic activity. The studies examined the effects ofL-685,458 on cleavage at the γ-secretase scissile bond of the substratesin both the cellular and in vitro assays.

[0095] L-685,458 is a putative γ-secretase inhibitor having thefollowing structure:

[0096] The effect of a γ-secretase inhibitor on γ-secretase activity wasmeasured using Chinese hamster ovary fibroblasts that stably expressAPP695 (CHO/APP695, provided by Dr. S. Sisodia (University of Chicago,Chicago, Ill.)). CHO/APP695 were grown in 90% DMEM, 10% fetal bovineserum, 2 mM glutamine, 100 μg/ml each of penicillin and streptomycin,and 0.2 mg/ml G418. CHO/APP695 cells were seeded in 96-well dishes at2×10⁴ cells/well. Aβ-peptide formation was detected by either ECL orusing radiolabeling.

[0097] For ECL detection, the media was replenished the next day with orwithout L-685,458. The Aβ(x-40) and Aβ(x-42) levels in the 24 hourconditioned media (CM) were measured by ECL.

[0098] For the radiolabeling experiments, CHO/APP695 cells (in 100 mmdishes) were grown to 70% confluency in complete media. Afterwards,these cells were cultured±1 μM L-685,458 for 24 hours, switched toMet-free medium (±1 μM L-685,458) for 20 minutes, pulsed with 1 mCi/mlof S³⁵-Met (Amersham Life Sciences, Inc, Arlington Heights, Ill.) inMet-free medium (±1 μM L-685,458) for 2 hours, and subsequently chasedin complete medium (±1 μM L-685,458) for 45 minutes. The cells werewashed with Hanks balanced salt solution and lysed with 1 ml of RIPA.The insoluble residue was removed by centrifugation. The 4G8 antibody(final concentration, 2 μg/ml) was added to the supernatant fractions.The samples were gently rotated overnight at 4° C. Protein G-agarosebeads (Pharmacia Biotech, Piscataway, N.J.) were added the next day, thesamples were rotated for an additional 2 hours and then centrifuged. Theprotein G-agarose beads from the 4G8 immunoprecipitation step werewashed 4 times with RIPA. An equal volume of SDS sample buffer (Novex,San Diego, Calif.) was added and the samples were boiled for 5 minutesand fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)using 16% polyacrylamide-Tricine gels (Novex). ¹⁴C-labeled proteinmarkers (Amersham Life Sciences) were co-electrophoresed. The gel wasdried and exposed to X-Omatic film (Kodak, Rochester, N.Y.) for 24-72hours.

[0099] Treatment of CHO/APP695 cells with L-685,458 results in theintracellular accumulation of APP immunoreactive fragments thatco-migrate with α-CTF and β-CTF (data not shown)—a result that isconsistent with γ-secretase inhibition. Moreover, L-685,458 blocked Aβ40and Aβ42 secretion from CHO/APP695 cells in a dose-dependent manner withIC₅₀ values for suppression of Aβ40 and Aβ42 secretion of 130 and 200nM, respectively.

[0100] Similarly, L-685,458 inhibits “solubilized γ-secretase” mediatedprocessing of SEQ. ID. NO. 9 substrate that results in the generation ofthe Aβ40- and Aβ42-related products. The IC₅₀ values for inhibition ofthe Aβ40 and Aβ42 cleavage events in the in vitro assay are bothapproximately 1 nM. All of the apparent “solubilized γ-secretase”activity is inhibited by L-685,458.

Example 6

[0101] Characterization of Product Formation

[0102] Incubation of HeLa cell membranes or “solubilized γ-secretase”with SEQ. ID. NO. 9 substrate generates M-Aβ42 and M-Aβ40. The Aβ42/Aβ40cleavage ratios, approximately 0.1, with both intact membranes and“solubilized γ-secretase” are similar to the corresponding ratio thathas been reported for Aβ peptides secreted into the CM of cultured cellsthat are overexpressing APP (Asami-Odaka et al., 1995. Biochemistry 34,10272-10278).

[0103] The apparent K_(m) value of “solubilized γ-secretase” for SEQ.ID. NO. 9 substrate (assaying for Aβ40 cleavage) is approximately 1 μM(FIG. 2A). Under the experimental conditions employed, the timedependence of product formation is linear over a 3 hour interval (FIG.2B). The production of the M-Aβ40 and M-Aβ42 are both blocked bypepstatin, a classical inhibitor of aspartyl class proteases (FIG. 2D).The IC₅₀ values of pepstatin for inhibiting the generation of the Aβ40and Aβ42 termini are 4.0 and 5.9 μM, respectively.

Example 7

[0104] pH Dependency

[0105] The pH dependence of in vitro γ-secretase activity was determinedusing pH-adjusted 50 mM MES and 50 mM PIPES for the pH 5.0-6.5 and pH6.5-9.0 ranges, respectively. The pH dependence of “solubilizedγ-secretase” activity showed a pH optimum of 7.0 (FIG. 2C).

Example 8

[0106] Detergent Effect

[0107] SEQ. ID. NO. 9 substrate (1.7 μM) was incubated with cellmembranes (0.5 mg/ml) in presence of CHAPSO, CHAPS or Triton X-100 (0,0.125, 0.25, 0.5, or 1%) in buffer B at 37° C. The results are shown inTable II. TABLE II M-Aβ 40 production (ECL signal × 10⁻⁶⁾ [Detergent](%) CHAPSO CHAPS Triton X-100 0 0.03 0.03 0.03 0.125 2.2 0.03 0.02 0.258.74 3.21 0.02 0.5 1.22 0.27 0.02 0.75 0.07 0.04 0.02 1 0.03 0.03 0.02

[0108] Simple addition of SEQ. ID. NO. 9 substrate to membranes preparedfrom HeLa cells does not lead to substrate cleavage and generation ofM-Aβ40. Inclusion of CHAPSO or CHAPS during the incubation of HeLa cellmembranes and SEQ. ID. NO. 9 substrate promotes cleavage at theγ-secretase scissile bond. Triton X-100 fails to potentiate in vitroγ-secretase activity. Optimal activity is observed in the presence of0.25% CHAPSO. Higher concentrations of CHAPSO lead to a progressivedecline in γ-secretase activity. Membranes prepared from human embryonickidney (HEK293) cells or Chinese hamster ovary (CHO) cells also processSEQ. ID. NO. 9 substrate in the presence of 0.25% CHAPSO to generateM-Aβ40 (data not shown).

[0109] Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1 18 1 100 PRT Artificial Sequence Beta-CTF domain 1 Met Asp Ala Glu PheArg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15 Lys Leu Val PhePhe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30 Ile Gly Leu MetVal Gly Gly Val Val Ile Ala Thr Val Ile Val Ile 35 40 45 Thr Leu Val MetLeu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60 Val Val Glu ValAsp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 75 80 Lys Met GlnGln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 90 95 Gln Met GlnAsn 100 2 100 PRT Artificial Sequence Beta-CTF domain 2 Met Asp Ala GluPhe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15 Lys Leu ValPhe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30 Ile Gly LeuMet Val Gly Gly Val Val Ile Ala Thr Val Val Val Ile 35 40 45 Thr Leu ValMet Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60 Val Val GluVal Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 75 80 Lys MetGln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 90 95 Gln MetGln Asn 100 3 100 PRT Artificial Sequence Beta-CTF domain 3 Met Asp AlaGlu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15 Lys LeuVal Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30 Ile GlyLeu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Ile Ile 35 40 45 Thr LeuVal Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60 Val ValGlu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 75 80 LysMet Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 90 95 GlnMet Gln Asn 100 4 100 PRT Artificial Sequence Beta-CTF domain 4 Met AspAla Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15 LysLeu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30 IleGly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Gly Ile 35 40 45 ThrLeu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60 ValVal Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 75 80Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 90 95Gln Met Gln Asn 100 5 100 PRT Artificial Sequence Beta-CTF domain 5 MetAsp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Phe Ile 35 40 45Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 7580 Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 9095 Gln Met Gln Asn 100 6 100 PRT Artificial Sequence Beta-CTF domain 6Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 1015 Lys Leu Val Phe Phe Gly Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 2530 Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile 35 4045 Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 5560 Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 7075 80 Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 8590 95 Gln Met Gln Asn 100 7 100 PRT Artificial Sequence Beta-CTF domain7 Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 1015 Lys Leu Val Phe Phe Ala Gln Asp Val Gly Ser Asn Lys Gly Ala Ile 20 2530 Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile 35 4045 Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 5560 Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 7075 80 Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 8590 95 Gln Met Gln Asn 100 8 8 PRT Artificial Sequence Hydrophilic moiety8 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 9 108 PRT Artificial SequenceGamma-secretase substrate 9 Met Asp Ala Glu Phe Arg His Asp Ser Gly TyrGlu Val His His Gln 1 5 10 15 Lys Leu Val Phe Phe Ala Glu Asp Val GlySer Asn Lys Gly Ala Ile 20 25 30 Ile Gly Leu Met Val Gly Gly Val Val IleAla Thr Val Ile Val Ile 35 40 45 Thr Leu Val Met Leu Lys Lys Lys Gln TyrThr Ser Ile His His Gly 50 55 60 Val Val Glu Val Asp Ala Ala Val Thr ProGlu Glu Arg His Leu Ser 65 70 75 80 Lys Met Gln Gln Asn Gly Tyr Glu AsnPro Thr Tyr Lys Phe Phe Glu 85 90 95 Gln Met Gln Asn Asp Tyr Lys Asp AspAsp Asp Lys 100 105 10 108 PRT Artificial Sequence Gamma-secretasesubstrate 10 Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His HisGln 1 5 10 15 Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys GlyAla Ile 20 25 30 Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val ValVal Ile 35 40 45 Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile HisHis Gly 50 55 60 Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg HisLeu Ser 65 70 75 80 Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr LysPhe Phe Glu 85 90 95 Gln Met Gln Asn Asp Tyr Lys Asp Asp Asp Asp Lys 100105 11 108 PRT Artificial Sequence Gamma-secretase substrate 11 Met AspAla Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15 LysLeu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30 IleGly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Ile Ile 35 40 45 ThrLeu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60 ValVal Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 75 80Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 90 95Gln Met Gln Asn Asp Tyr Lys Asp Asp Asp Asp Lys 100 105 12 108 PRTArtificial Sequence Gamma-secretase substrate 12 Met Asp Ala Glu Phe ArgHis Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15 Lys Leu Val Phe PheAla Glu Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30 Ile Gly Leu Met ValGly Gly Val Val Ile Ala Thr Val Ile Gly Ile 35 40 45 Thr Leu Val Met LeuLys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60 Val Val Glu Val AspAla Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 75 80 Lys Met Gln GlnAsn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 90 95 Gln Met Gln AsnAsp Tyr Lys Asp Asp Asp Asp Lys 100 105 13 108 PRT Artificial SequenceGamma-secretase substrate 13 Met Asp Ala Glu Phe Arg His Asp Ser Gly TyrGlu Val His His Gln 1 5 10 15 Lys Leu Val Phe Phe Ala Glu Asp Val GlySer Asn Lys Gly Ala Ile 20 25 30 Ile Gly Leu Met Val Gly Gly Val Val IleAla Thr Val Ile Phe Ile 35 40 45 Thr Leu Val Met Leu Lys Lys Lys Gln TyrThr Ser Ile His His Gly 50 55 60 Val Val Glu Val Asp Ala Ala Val Thr ProGlu Glu Arg His Leu Ser 65 70 75 80 Lys Met Gln Gln Asn Gly Tyr Glu AsnPro Thr Tyr Lys Phe Phe Glu 85 90 95 Gln Met Gln Asn Asp Tyr Lys Asp AspAsp Asp Lys 100 105 14 108 PRT Artificial Sequence Gamma-secretasesubstrate 14 Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His HisGln 1 5 10 15 Lys Leu Val Phe Phe Gly Glu Asp Val Gly Ser Asn Lys GlyAla Ile 20 25 30 Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val IleVal Ile 35 40 45 Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile HisHis Gly 50 55 60 Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg HisLeu Ser 65 70 75 80 Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr LysPhe Phe Glu 85 90 95 Gln Met Gln Asn Asp Tyr Lys Asp Asp Asp Asp Lys 100105 15 108 PRT Artificial Sequence Gamma-secretase substrate 15 Met AspAla Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln 1 5 10 15 LysLeu Val Phe Phe Ala Gln Asp Val Gly Ser Asn Lys Gly Ala Ile 20 25 30 IleGly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile 35 40 45 ThrLeu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly 50 55 60 ValVal Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser 65 70 75 80Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu 85 90 95Gln Met Gln Asn Asp Tyr Lys Asp Asp Asp Asp Lys 100 105 16 32 DNAArtificial Sequence Primer 16 ggaattccat atggatgcag aattccgaca tg 32 1761 DNA Artificial Sequence Primer 17 cgcggatccc tatttatcgt catcgtctttgtagtcgttc tgcatctgct caaagaactt 60 g 61 18 327 DNA Artificial SequenceDNA Encoding SEQ. ID. NO. 9 18 atggatgcag aattccgaca tgactcaggatatgaagttc atcatcaaaa attggtgttc 60 tttgcagaag atgtgggttc aaacaaaggtgcaatcattg gactcatggt gggcggtgtt 120 gtcatagcga cagtgatcgt catcaccttggtgatgctga agaagaaaca gtacacatcc 180 attcatcatg gtgtggtgga ggttgacgccgctgtcaccc cagaggagcg ccacctgtcc 240 aagatgcagc agaacggcta cgaaaatccaacctacaagt tctttgagca gatgcagaac 300 gactacaaag acgatgacga taaatag 327

What is claimed is:
 1. A γ-secretase substrate consisting of: a) a β-CTFdomain; and b) a hydrophilic polypeptide moiety covalently joined to thecarboxyl terminus of said β-CTF domain.
 2. The substrate of claim 1,wherein said β-CTF domain is substantially similar to SEQ. ID. NO.
 1. 3.The substrate of claim 2, wherein said β-CTF domain consists essentiallyof a sequence selected from the group consisting of: SEQ. ID. NO. 1,SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID.NO. 6, and SEQ. ID. NO.
 7. 4. The substrate of claim 3, wherein saidhydrophilic polypeptide moiety is about 5 to about 15 amino acids inlength and contains a net charge that is greater than ±2 (absolutevalue).
 5. The substrate of claim 4, where said hydrophilic moiety isabout 8 amino acids in length and contains a net charge that is greaterthan −2 (absolute value).
 6. The substrate of claim 5, wherein saidβ-CTF domain consists of a sequence selected from the group consistingof: SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ.ID. NO. 5, SEQ. ID. NO. 6, and SEQ. ID. NO.
 7. 7. The substrate of claim1, wherein said substrate is substantially similar to SEQ. ID. NO.
 9. 8.The substrate of claim 7, wherein said substrate consists of a sequenceselected from the group consisting of: SEQ. ID. NO. 9, SEQ. ID. NO. 10,SEQ. ID. NO. 11, SEQ. ID. NO. 12, SEQ. ID. NO. 13, SEQ. ID. NO. 14, andSEQ. ID. NO.
 15. 9. The substrate of claim 8, wherein said substrateconsists of SEQ. ID. NO.
 9. 10. A nucleic acid comprising a nucleotidebase sequence encoding for the substrate of claim
 1. 11. The nucleicacid of claim 10, wherein said nucleic acid is an expression vector. 12.A recombinant cell comprising the nucleic acid of claim
 10. 13. A methodfor assaying γ-secretase activity comprising the step of measuringcleavage of the substrate of any one of clams 1-9 by γ-secretase in thepresence of an effective amount of a zwitterionic detergent.
 14. Themethod of claim 13, wherein said zwitterionic detergent is either CHAPSor CHAPSO.
 15. The method of claim 14, wherein said effective amount isabout 0.25%.
 16. The method of claim 15, wherein said measuringcomprises the use of an antibody that binds to the carboxyl terminus ofthe Aβ peptide-related product produced by said cleavage.
 17. The methodof claim 16, wherein said method is performed in the presence of one ormore compounds that inhibit γ-secretase activity.
 18. A method formeasuring the ability of a compound to affect γ-secretase activitycomprising the steps of: a) combining together the substrate of any oneof clams 1-9, said compound, and a preparation comprising γ-secretaseactivity, under reaction conditions allowing for γ-secretase activity,wherein said reaction conditions comprise an effective amount of azwitterionic detergent; and b) measuring γ-secretase activity.
 19. Themethod of claim 18, wherein said zwitterionic detergent is either CHAPSor CHAPSO.